Fluorescent polarization assays involving multivalent metal ions and systems

ABSTRACT

Methods, systems, kits for carrying out a wide variety of different assays that comprise providing a first reagent mixture which comprises a first reagent having a fluorescent label. A second reagent is introduced into the first reagent mixture to produce a second reagent mixture, where the second reagent reacts with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent. A polyion is introduced into at least one of the first and second reagent mixtures, and the fluorescent polarization in the second reagent mixture relative to the first reagent mixture is determined, this fluorescent polarization being indicative of the rate or extent of the reaction.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.09/316,447, filed May 21, 1999, now U.S. Pat. No. 6,287,774, which isincorporated herein in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Virtually all chemical, biological and biochemical research depends uponthe ability of the investigator to determine the direction of herresearch by assaying reaction mixtures for the presence or absence of aparticular chemical species within the reaction mixture. In a simplecase, the rate or efficiency of a reaction is assayed by measuring therate of production of the reaction product, or the depletion of areaction substrate. Similarly, interactive reactions, e.g., binding ordissociation reactions are generally assayed by measuring the amount ofbound or free material in the resultant reaction mixture.

For certain reactions, the species of interest, or a suitable surrogate,is readily detectable and distinguishable from the remainder of thereagents. Thus, in order to detect such species, one merely needs tolook for it. Often, this is accomplished by rendering a reaction productoptically detectable and distinguishable from the reagents by virtue ofan optical signaling element or moiety that is only present or active onthe product or the substrate. By measuring the level of optical signal,one can directly ascertain the amount of product or remaining substrate.

Unfortunately, many reactions of particular interest do not have thebenefit of having a readily available surrogate reagent that producessignal only when subjected to the reaction of interest. For example,many reactions that are of great interest to the biological researchfield do not subject their reagents to the types of modifications thatcan give rise to substantial optical property changes. Researchers haveattempted to engineer substrates, which give rise to optical propertychanges. For example, typical binding reactions between two moleculesresult in a bound complex of those molecules. However, even when onemember of the binding pair is labeled, the formation of the complex doesnot generally give rise to an optically detectable difference betweenthe complex and the labeled molecule. As a result, most binding assaysrely upon the immobilization of one member or molecule of the bindingpair. The labeled molecule is then contacted with the immobilizedmolecule, and the immobilizing support is washed. Following washing, thesupport is then examined for the presence of the labeled molecule,indicating binding of the labeled component to the unlabeled,immobilized component. Vast arrays of different binding member pairs areoften prepared in order to enhance the throughput of the assay format.See, e.g., U.S. Pat. No. 5,143,854 to Pirrung et al.

Alternatively, in the case of nucleic acid hybridization assays,researchers have developed complementary labeling systems that takeadvantage of the proximity of bound elements to produce fluorescentsignals, either in the bound or unbound state. See, e.g., U.S. Pat. Nos.5,688,648, 5,707,804, 5,728,528, 5,853,992, and 5,869,255 to Mathies etal. for a description of FRET dyes, and Tyagi et al. Nature Biotech.14:303-8 (1996), and Tyagi et al., Nature Biotech. 16:49-53 (1998) for adescription of molecular beacons.

As noted above, binding reactions are but one category of assays thatgenerally do not produce optically detectable signals. Similarly, thereare a number of other assays whose reagents and/or products cannot bereadily distinguished from each other, even despite the incorporation ofoptically detectable elements. For example, kinase assays thatincorporate phosphate groups onto phosphorylatable substrates do notgenerally have surrogate substrates that produce a detectable signalupon completion of the phosphorylation reaction. Instead, such reactionstypically rely upon a change in the structure of the product, whichstructural change is used to separate the reactants from the product.The separated product is then detected. As should be apparent, assaysrequiring additional separation steps can be extremely time consumingand less efficient, as a result of losses during the various assaysteps.

It would generally be desirable to be able to perform theabove-described assay types without the need for solid supports,additional separation steps, or the like. The present invention meetsthese and a variety of other important needs.

SUMMARY OF THE INVENTION

The present invention provides methods, systems, kits and the like forcarrying out a wide variety of different assays. These assays typicallycomprise providing a first reagent mixture which comprises a firstreagent having a fluorescent label. A second reagent is introduced intothe first reagent mixture to produce a second reagent mixture, where thesecond reagent reacts with the first reagent to produce a fluorescentlylabeled product having a substantially different charge than the firstreagent. A polyion is introduced into at least one of the first andsecond reagent mixtures, and the fluorescent polarization in the secondreagent mixture relative to the first reagent mixture is determined,this fluorescent polarization being indicative of the rate or extent ofthe reaction.

Another aspect of the present invention is a method of detecting areaction. The method comprises providing a first reagent mixture whichcontains a first reagent having a fluorescent label. A second reagent isintroduced into the first reagent mixture to produce a second reagentmixture. The second reagent reacts with the first reagent to produce afluorescently labeled product having a substantially different chargethan the first reagent. A polyion is introduced into at least one of thefirst and second reagent mixtures and fluorescent polarization iscompared in the second reagent mixture relative to the first reagentmixture.

A further aspect of the present invention is a method of identifying thepresence of a subsequence of nucleotides in a target nucleic acid. Themethod comprises contacting the target nucleic acid sequence with anuncharged, fluorescently labeled nucleic acid analog in a first reactionmixture. The nucleic acid analog is complementary to the subsequencewhereby the nucleic acid analog is capable of specifically hybridizingto the subsequence to form a first hybrid. The first reaction mixture iscontacted with a polyion and the level of fluorescence polarization ofthe first reaction mixture in the presence of the polyion is compared tothe level of fluorescence polarization of the uncharged nucleic acidanalog in the absence of the target nucleic acid sequence. An increasein the level of fluorescence polarization indicates the presence of thefirst hybrid.

Another aspect of the present invention is a method of detecting thephosphorylation of a phosphorylatable compound. The method comprisesproviding the phosphorylatable compound with a fluorescent label. Thephosphorylatable compound is contacted with a kinase enzyme in thepresence of a phosphate group in a first mixture and then contacting thefirst mixture with a polyion. The level of fluorescence polarizationfrom the first mixture in the presence of the polyion is compared to thelevel of fluorescence polarization from the phosphorylatable compoundwith the fluorescent label in the absence of the kinase enzyme.

A further aspect of the present invention is a method of detecting thephosphorylation of a phosphorylatable compound. The method comprisesproviding the phosphorylatable compound with a fluorescent label. Thephosphorylatable compound is contacted with a kinase enzyme in thepresence of a phosphate group in a first mixture. The first mixture iscontacted with a second reagent mixture comprising a protein having achelating group associated therewith, and a metal ion selected fromFe³⁺, Ca²⁺, Ni²⁺ and Zn²⁺. The level of fluorescence polarization fromthe first mixture in the presence of the second mixture is compared tothe level of fluorescence polarization from the phosphorylatablecompound with the fluorescent label in the absence of the kinase enzyme.

A further aspect of the present invention is an assay system comprisinga fluid receptacle. The system contains a first reaction zone containinga first reagent mixture which comprises a first reagent having afluorescent label, a second reagent that reacts with the first reagentto produce a fluorescently labeled product having a substantiallydifferent charge than the first reagent, and a polyion. The system alsoincludes a detection zone and a detector disposed in sensorycommunication with the detection zone. The detector is configured todetect the level of fluorescence polarization of reagents in thedetection zone.

Another aspect of the present invention is an assay system comprising afirst channel disposed in a body structure. The first channel is fluidlyconnected to a source of a first reagent mixture which comprises a firstreagent having a fluorescent label, a source of a second reagent thatreacts with the first reagent to produce a fluorescently labeled producthaving a substantially different charge than the first reagent; and asource of a polyion. The system also includes a material transportsystem for introducing the first reagent, the second reagent and thepolyion into the first channel and a detector disposed in sensorycommunication with the first channel. The detector is configured todetect the level of fluorescence polarization of reagents in thedetection zone.

Another aspect of the present invention is a kit. The kit includes avolume of a first reagent which comprises a fluorescent label; a volumeof a second reagent which reacts with the first reagent to produce afluorescent product having a substantially different charge from thefirst reagent; and a volume of a polyion. The kit also includesinstructions for determining the level of fluorescence polarization ofthe first reagent, mixing the first reagent, the second reagent and thepolyion in a first mixture, determining the level of fluorescencepolarization of the first mixture, and comparing the level fluorescencepolarization of the first reagent to the level of fluorescencepolarization of the first mixture.

Another aspect of the present invention is an assay system forquantifying a reaction parameter which comprises providing a firstreagent mixture. The first reagent mixture includes a first reagenthaving a fluorescent label. A second reagent is introduced into thefirst reagent mixture to produce a second reagent mixture. The secondreagent reacts with the first reagent to produce a fluorescently labeledproduct having a substantially different charge than the first reagent.A polyion is introduced into at least one of the first and secondreagent mixtures. The system also includes a computer implementedprocess, comprising the steps of determining a first level offluorescence polarization of the first reagent mixture; determining asecond level of fluorescence polarization of the second reagent mixture;comparing the first and second levels of fluorescent polarization; andcalculating the reaction parameter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration of one embodiment of a general assayprocess performed in accordance with the present invention.

FIG. 2 is a schematic illustration of a binding assay, e.g., a nucleicacid hybridization assay, performed in accordance with the presentinvention.

FIG. 3 is a schematic illustration of an enzyme assay, e.g., a kinaseassay, performed in accordance with the present invention.

FIG. 4 is a schematic illustration of a phosphatase assay performed inaccordance with the present invention.

FIG. 5 is a general schematic illustration of an overall system used tocarry out the assay methods of the present invention.

FIG. 6 is a schematic illustration of a multi-layered microfluidicdevice that is optionally employed as a reaction/assay receptacle in thepresent invention.

FIG. 7 is a schematic illustration of a microfluidic deviceincorporating an external sampling pipettor as a reaction/assayreceptacle in the present invention.

FIG. 8 is a schematic illustration of one example of an opticaldetection system for use with the present invention.

FIG. 9 is a flow chart of a software program or computer implementedprocess carried out by an assay system in performing the assays of thepresent invention.

FIG. 10A illustrates an exemplary computer system that may be used toexecute software for use in practicing the methods of the invention.

FIG. 10B illustrates a block diagram of the architecture of the computersystem of FIG. 10A.

FIG. 11 illustrates the interfacing of a microfluidic device with otherelements of a system for controlling material movement, detecting assayresults from the microfluidic device, and analyzing those results.

FIG. 12 is a plot of fluorescent polarization of a fluorescentphosphorylated compound in the presence of increasing amounts of apolycation.

FIG. 13 is a plot of fluorescent polarization of a mixture offluorescent phosphorylatable substrate and phosphorylated product, wherethe relative concentrations of substrate and product are varied, in theadditional presence of a polycation.

FIG. 14 is a similar plot to that shown in FIG. 13, except utilizing adifferent phosphorylatable substrate and phosphorylated product.

FIG. 15 is a bar graph of the fluorescent polarization level of afluorescent PNA probe used to interrogate a non-complementary andcomplementary target DNA sequence, in the absence and the presence ofvarying levels of polycation.

FIG. 16 is a plot of fluorescent polarization of a mixture of a targetnucleic acid sequence and a perfectly complementary fluorescent PNAprobe (upper line, diamonds) and a mixture of a target sequence that iscomplementary but for a single base mismatch with a fluorescent PNAprobe (lower line, squares).

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention generally provides assay methods and systems whichare broadly useful in a variety of different contexts where othertypical assay formats cannot be used. The present methods and systemsare capable of detecting a reaction product in the presence of thereaction substrate, despite the fact that the product is detected byvirtue of a property that it shares with the reaction substrate, e.g., afluorescent labeling group.

In general, the methods and systems of the present invention distinguishreaction product from a reaction substrate by virtue of a change in thelevel of charge between the two as a result of the reaction. The chargeon one of these reaction components, whether it is located on thesubstrate or the product, is used to associate that component with arelatively large polyionic compound. The preferential association of thelarge polyionic compound with either the substrate or the productresults in a substantial difference in the level of polarization offluorescent emissions from that component when it is excited usingpolarized light. Because the large compound associates preferentiallywith only one of the substrate or product, as a result of theavailability or elimination of charge due to the reaction of interest,that association and its consequent change in fluorescence polarizationbecomes an indicator of the progress of the reaction of interest.

II. Assay Methods

In at least one aspect, the present invention provides a method ofdetecting a chemical, biochemical or biological reaction. The methodcomprises providing a first reagent mixture, which comprises a firstreagent having a fluorescent label. A second reagent is introduced intothe first reagent mixture to produce a second reagent mixture. Thesecond reagent is generally capable of reacting or otherwise interactingwith the first reagent to produce a fluorescently labeled product havinga substantially different charge than the first reagent. As used herein,the phrase “substantially different charge” means that the net charge onthe product differs from that of the first reagent by an amountsufficient to permit the differential association of the substrate andproduct with a polyionic compound as described herein. This differentialcharge may be a fraction of a charge, on average over the entire reagentmolecule. However, in preferred aspects, the substrate and producttypically differ by at least one charge unit at the pH at which theassay is being performed. For example, a product that bears a net singlepositive or negative charge has a substantially different charge from afirst reagent that has a net neutral charge, as the phrase is usedherein. Similarly, a product that bears two positive charges has asubstantially different charge from a first reagent that has a singlepositive charge. Preferably, a product having a substantially differentcharge than the first reagent will differ in net charge by at least twocharge units, and in certain aspects, many more than two charge unitsdifference, e.g., in the nucleic acid applications described in greaterdetail below.

A polyion is contacted with either or both of the first and secondreagent mixtures, depending upon the assay type that is being performed,and the nature of the product produced by the reaction of interest.Because the reaction of interest produces a product with a substantiallydifferent charge than the substrate, that product will interact quitedifferently with the polyion than will the substrate, e.g., increased ordecreased interaction/association. The association or lack ofassociation has a profound effect on the ability of the product todepolarize emitted fluorescence. Specifically, and as described ingreater detail below, the relatively small first reagent has arelatively fast rotational diffusion rate. This rotational diffusionrate is responsible for a fluorescent compound's ability to emitdepolarized fluorescence when excited with polarized light. However,association of a large polyionic compound with a small fluorescentmolecule will significantly slow the rate of rotational diffusion ofthat molecule, and reduce its ability to emit depolarized fluorescence.

The level of fluorescent polarization in the second reagent mixture isthen compared to the level of fluorescent polarization from the firstreagent mixture. By comparing these values, one can quantify the amountof fluorescence that is emitted from material bound to the polyion. Asdescribed in greater detail below, the assay is adjusted, e.g., byadjusting pH, ionic strength or the like, so that only one of the firstreagent or product is capable of associating with the polyion. Thus, thechange in fluorescence polarization is used to calculate a quantitativemeasure of the amount of product produced or first reagent consumed, andtherefore becomes a measure of the reaction.

The principles behind the use of fluorescence polarization measurementsas a method of measuring binding among different molecules arerelatively straight-forward. Briefly, when a fluorescent molecule isexcited with a polarized light source, the molecule will emitfluorescent light in a fixed plane, e.g., the emitted light is alsopolarized, provided that the molecule is fixed in space. However,because the molecule is typically rotating and tumbling in space, theplane in which the fluoresced light is emitted varies with the rotationof the molecule (also termed the rotational diffusion of the molecule).Restated, the emitted fluorescence is generally depolarized. The fasterthe molecule rotates in solution, the more depolarized it is.Conversely, the slower the molecule rotates in solution, the lessdepolarized, or the more polarized it is. The polarization value (P) fora given molecule is proportional to the molecule's “rotationalcorrelation time,” or the amount of time it takes the molecule to rotatethrough an angle of 57.3° (1 radian). The smaller the rotationalcorrelation time, the faster the molecule rotates, and the lesspolarization will be observed. The larger the rotational correlationtime, the slower the molecule rotates, and the more polarization will beobserved. Rotational relaxation time is related to viscosity (η),absolute temperature (T), molar volume (V), and the gas constant (R).The rotational correlation time is generally calculated according to thefollowing formula:

Rotational Correlation Time=3ηV/RT  (1)

As can be seen from the above equation, if temperature and viscosity aremaintained constant, then the rotational relaxation time, and therefore,the polarization value, is directly related to the molecular volume.Accordingly, the larger the molecule, the higher its fluorescentpolarization value, and conversely, the smaller the molecule, thesmaller its fluorescent polarization value.

In the performance of fluorescent binding assays, a typically small,fluorescently labeled molecule, e.g., a ligand, antigen, etc., having arelatively fast rotational correlation time, is used to bind to a muchlarger molecule, e.g., a receptor protein, antibody etc., which has amuch slower rotational correlation time. The binding of the smalllabeled molecule to the larger molecule significantly increases therotational correlation time (decreases the amount of rotation) of thelabeled species, namely the labeled complex over that of the freeunbound labeled molecule. This has a corresponding effect on the levelof polarization that is detectable. Specifically, the labeled complexpresents much higher fluorescence polarization than the unbound, labeledmolecule.

Generally, the fluorescence polarization level is calculated using thefollowing formula:

P=[I(∥)−I(⊥)]/[I(∥)+I(⊥)]  (2)

Where I(∥) is the fluorescence detected in the plane parallel to theexcitation light, and I(⊥) is the fluorescence detected in the planeperpendicular to the excitation light.

In performing screening assays, e.g., for potential inhibitors,enhancers, agonists or antagonists of the binding function in question,the fluorescence polarization of the reaction mixture is compared in thepresence and absence of different compounds, to determine whether thesedifferent compounds have any effect on the binding function of interest.In particular, in the presence of inhibitors of the binding function,the fluorescence polarization will decrease, as more free, labeledligand is present in the assay. Conversely, enhancers of the bindingfunction will result in an increase in the fluorescent polarization, asmore complex and less free-labeled-ligand are present in the assay.

As noted above, the assay methods of the present invention typicallyutilize a first reagent that includes a fluorescent labeling group. Thenature of the first reagent generally depends upon the type of assaythat is being performed. Typically, the assays that may be performedutilizing the present invention are myriad, including a wide range ofbinding or other associative assays, as well as assays of enzymaticactivity. Accordingly, the first reagents, as described herein,generally include, e.g., one member of a specific binding pair, i.e.,antibody/antigen pairs, receptor/ligand pairs, complementary nucleicacids or analogs thereof, binding proteins and their binding sites.Alternatively, or additionally, the first reagent may comprise asubstrate which is modified by the reaction of interest, e.g., byaddition to, subtraction from or alteration of the chemical structure ofthe first reagent. Some specific examples of such substrates include,e.g., kinase substrates which include phosphorylatable moieties, e.g.,serine, threonine and tyrosine phosphorylation sites, and the like,phosphorylated substrates for phosphatase enzymes, amino or ketocontaining substrates subject to amino transferases, alcohols convertedto carboxyls (e.g., via glucose-6-phosphate dehydrogenase), as well assubstrates for: sulfatases; phosphorylases; esterases; hydrolases (e.g.,proteases); oxidases, and the like.

The first reagent may be charged, either positively or negatively, or itmay be neutral, depending upon the nature of the assay that is to beperformed. The fluorescent label on the first reagent may be selectedfrom any of a variety of different fluorescent labeling compounds.Generally, such fluorescent labeling materials are commerciallyavailable from, e.g., Molecular Probes (Eugene, Oreg.). Typically,fluorescein or rhodamine derivatives are particularly well suited to theassay methods described herein. These fluorescent labels are coupled tothe first reagent, e.g., covalently through well known couplingchemistries. For a discussion of labeling groups and chemistries, see,e.g., Published International Patent Application No. WO 98/00231, whichis incorporated herein by reference.

Also as noted above, the second reagent generally reacts, interacts orotherwise associates or binds with the first reagent to produce afluorescent product that includes a substantially different charge thanthe first reagent. As with the first reagent, this second reagentoptionally comprises one member of a specific binding pair, e.g., themember that is complementary to the first reagent, provided that thehybrid of the two members of the binding pair, or first and secondreagents, bears a charge that is substantially different from the chargeof the first reagent member of the binding pair. In many cases, thisinvolves a second reagent that is charged while the first reagent isneutral, or a second reagent that is highly charged as compared to afirst reagent that is only moderately charged. Alternatively, theassociation of the first and second reagents confers a conformationalchange that yields a charged product, or binds to and masks chargedresidues on the first reagent.

Because the product produced by the interaction of the first and secondreagents has a different charge than the first reagent alone, it willinteract differently with other charged molecules. In particular,polyionic compounds that bear a substantial number of charges willgenerally interact with charged materials in a charge dependent manner.In the case of the present invention, large polyionic compounds are usedin order to “tag” the product (or in some cases, the first reagent) witha relatively large compound that will affect the product's ability toemit depolarized fluorescence.

Preferred polyions for use in the present invention include polyaminoacids, e.g., proteins, polypeptides, i.e., polylysine, polyhistidine,and polyarginine. Other polyions that are useful in accordance with theinvention include organic polyions, i.e., polyacrylic acid,polycarboxylic acids, polyamines (e.g., polyethylamine), polysulfonicacids (e.g., polystyrene sulfonic acid) polyphosphoric acid (e.g.,polyvinylphosphoric acid), or copolymers of any or all of these, e.g.,mixed polymers of these polyamino acids, and the like. These polyionsare typically relatively large in comparison to the first and/or secondreagents, and/or the product that is being used in the assay ofinterest. As such, the size of the polyion may vary depending upon thesize of the first and/or second reagents and/or the product. Typically,the polyion will range in size from about 5 kD to about 1000 kD, andpreferably, from about 10 kD to about 100 kD. In the case of polyaminoacids, this typically constitutes a polymer of from about 50 to about10,000 amino acid monomers in length, and preferably from about 100 toabout 1000 monomers in length.

The polyions used in accordance with the present invention are generallycapable of interacting with the other components of the reaction mixturein a non-specific charge dependent manner. As a non-specificinteraction, it will be appreciated that the polyions used in accordancewith the present invention do not require the presence of a specificrecognition site in the product (or substrate). This non-specificinteraction thus provides for a broader applicability for the assays ofthe present invention. Also as noted, the polyion interacts with theproduct (or substrate) is a charge dependent fashion. As a result, inthe case of titratable polyions, this can require buffer conditions thatpermit the presence of the charges used in that interaction. Typically,polyionic materials preferably will have an isoelectric point (pI) thatprovides a significant level of charge at the relevant pH level for theassay conditions. Typically, buffers in which the assays of the presentinvention are carried out will typically be in the physiologicallyrelevant range, e.g., from about pH 6 to about pH 8, at which, thepolyionic compounds will have a sufficient charge level to interact withcharged reagents. However, as will be appreciated, one can generallyadjust the level of charge, and thus the level of interaction between apolyion and a product (or substrate) by adjusting the buffer in whichthese elements are disposed, thereby effecting the charge level of oneor both of the polyion or the product. Routine reaction tuning can alsobe used to optimize any given assay to yield optimal reaction rates aswell as interaction between the polyionic component and the product (orsubstrate).

The differential interaction between the polyion and the fluorescentproduct, as compared to its interaction with the fluorescent firstreagent is then used as a means for comparing the amount of productproduced. Specifically, a relatively small fluorescent compound, e.g.,the first reagent, generally emits relatively depolarized fluorescencewhen it is excited by polarized excitation light. This is generally dueto the faster rotational diffusion or “spin” of these smaller compounds.Larger compounds, on the other hand have slower spin and thus are morelikely to emit relatively polarized fluorescence when excited by apolarized excitation light source. By tagging the product with a large“label” in the form of a polyion, one substantially alters the product'sability to emit depolarized fluorescence. This property is then detectedand quantified as a measure of the reaction of the first and secondreagents. Typically, the detected fluorescence polarization, or P value,provides a measure of the ratio of bound label to free label, althoughassay results may also be determined as a difference betweenpre-reaction fluorescence polarization and post-reaction fluorescencepolarization, with the difference being an indication of the reaction'srate and/or completeness.

FIG. 1 schematically and generally illustrates the assay methods of thepresent invention. These illustrations are for example purposes only andare not intended to imply limits to the present invention. Briefly, asshown in FIG. 1, a fluorescently labeled first reagent 102 is provided.The first reagent has a relatively fast rotational diffusion rate. Thefirst reagent 102 is contacted with a second reagent, e.g., Enzyme I,which either mediates addition of or itself constitutes a charged group104 that associates with the first reagent 102 to create a chargedproduct 106. The resulting charged product typically has a rotationaldiffusion rate that is not substantially different from that of thefirst reagent.

The product is then contacted with a relatively large polyion 108, whichassociates with the charged fluorescent product 106. The resultingpolyion/charged fluorescent product 110 has a substantially reducedrotational diffusion rate as compared to the original first reagent. Asdescribed above, this difference in rotational diffusion rate isquantitatively measurable using, e.g., fluorescent polarizationdetection methods.

Although generally described in terms of the polyion associating withthe product of the reaction of interest, the methods described hereinalso operate in the reverse direction. Specifically, the first reagentoptionally is associated with the polyion, with all of thecharacteristics that entails. The reaction of interest then alters thecharge of the first reagent in producing a product. The product then hasa reduced or eliminated interaction with the polyion as compared to thefirst reagent. This reduced interaction then gives rise to a change inthe product's ability to emit polarized fluorescence relative to thefirst reagent. In some cases, this may require that the assay utilize aheterogeneous format, e.g., introducing the polyion after the reactionof interest is carried out, as a result of potential interfering effectsof the polyion on the reaction of interest. Methods of performing theassay methods of the invention in both homogeneous and heterogeneousformats are described in greater detail below.

The level of fluorescence polarization of the product then provides anindication of the amount of the fluorescent label that is bound to thepolyion, e.g., as the ratio of bound to free label. Typically,fluorescence polarization data are generally reported as the ratio ofthe difference of parallel and perpendicular fluorescence emissions tothe sum of these fluorescent emissions. Thus, the smaller the differencebetween these fluorescence emissions, e.g., the more depolarized theemissions, the smaller the polarization value. Conversely, morepolarized emissions yield larger numbers. As alluded to above, incomparing assay results, the polarization value (P) for the reaction mixis determined. The fraction of bound fluorescence, e.g., associated withthe polyion, is determined as:

F _(b)=(P−P _(f))/(P _(b) −P _(f))  (3)

where P_(b) is the P value of the bound species, and P_(f) is the Pvalue of the free species. Thus, the polarization value can be used asan absolute quantitative measurement of the ratio of product tosubstrate, where one has determined or is already aware of the P valuefor completely bound label and completely free label. Alternatively, asnoted above, one can measure the pre-reaction and post reactionfluorescence polarization, using the difference between the two as anindication of the amount of product produced. As noted above, the assaymethods also works for the inverted assay format, e.g., where thepolyion is associated with the first reagent, but not the fluorescentproduct. In this case, the difference between the fluorescentpolarization of the first reagent and the product is determined.

The P value serves as an indicator of the reaction of interest, e.g., byindicating the amount of product produced. As will be discussed ingreater detail below, once an assay reaction is quantifiable, one canuse that assay in a number of different applications, including forexample diagnostics, but particularly for screening of potentialinhibitors or enhancers of the reaction of interest. This is typicallyuseful in screening compound libraries against pharmacologicallyrelevant targets that utilize one or more of the reactions describedherein, e.g., binding, enzymatic modification and the like.

Although generally described in terms of detection of fluorescentpolarization, it will be readily appreciated that a variety of detectionschemes may be employed which detect the rate of rotation of a moleculeor the translation or lateral diffusion of a molecule that relates tothe size of the molecule. Examples of methods of detecting a molecule'srotation include, e.g., nuclear magnetic resonance spectroscopy,electron spin resonance spectroscopy, and triplet state absorbanceanisotropy. Examples of methods of detecting the translation rate ofmolecules include, e.g., fluorescent correlation spectroscopy,fluorescence recovery after photobleaching, and magnetic resonance spinexchange spectroscopies.

As noted repeatedly above, the general methods and systems of thepresent invention can be used in assaying a variety of different typesof biologically or biochemically relevant reactions, including enzymemediated reactions, binding reactions and hybridization reactions. Inthe case of binding reactions, the first reagent that bears afluorescent label is contacted with a second reagent that binds to thefirst reagent to yield a fluorescently labeled product. The secondreagent typically includes a level of charge such that the productresulting from the binding of the second reagent to the first reagenthas a charge that is substantially different than the first reagentalone.

A simple example of such a binding assay is a nucleic acid hybridizationassay. Specifically, in determining the presence of a particular nucleicacid sequence or subsequence in a sample or target nucleic acid, oneoften interrogates the target nucleic acid with shorter nucleic acidprobes that have a nucleotide sequence that is complementary to, andthus is capable of hybridizing to the sequence or subsequence ofinterest in the target. If the probe hybridizes to the target sequence,the presence of the subsequence of interest is indicated. Previouslydescribed high throughput methods have generally required that at leastone of the probe or the target sequence is immobilized, e.g., on a solidsupport or in a particular position in an oligonucleotide array (See,e.g., U.S. Pat. Nos. 5,143,854 and 5,744,305). While some solution basedhybridization detection methods have been described, these typicallyrequire specially synthesized reagents for the sequence that is to beinterrogated, e.g., including FRET dye pairs, molecular beacons, or thelike.

In the case of the present invention, the first reagent is typically anuncharged or positively charged nucleic acid analog, which bears afluorescent label. Suitable nucleic acid analogs are generally known andinclude, e.g., peptide nucleic acids (PNAs), methyl phosphonate polymersand cationic nucleic acid analogs. Because these nucleic acid analogsare neutral, or in some cases positively charged, they do not form acharge based association with the polycation component of the assay.

Because nucleic acids are highly charged species, an uncharged orpositively charged nucleic acid analog is used as the first reagent.This permits differentiation between the free probe and the probe thatis hybridized to the target sequence by virtue of the charge on thehybrid from the presence of the target sequence. Although the polyionwill associate with all of the target sequence, including that whichdoes not hybridize to the probe, that interaction is invisible to theinvestigator, as the result of that interaction not bearing afluorescent label. This nucleic acid hybridization assay isschematically illustrated in FIG. 2.

As shown, a target nucleic acid 202 (schematically illustrated) isinterrogated with a fluorescent probe 204 (the fluorescent label isindicated as an *), which typically comprises an uncharged nucleic acidanalog, e.g., a PNA probe. The probe is selected to be complementary toa particular nucleotide sequence, e.g., the sequence of interest, suchthat the probe will selectively hybridize to that sequence if it ispresent in the target nucleic acid 202. In its individual form, theprobe will have a relatively high rate of rotational diffusion due toits small size, as schematically illustrated by arrow 206, therebyemitting more highly depolarized fluorescence.

The reaction illustrated in panel I illustrates the case where thetarget sequence 202 contains the sequence of interest, so that the probe204 will hybridize to the target sequence 202 to form a first hybrid208. Due to the larger size of the hybrid relative to that of the probe,this hybridization reaction will result in a reduction in the rotationalrate of diffusion of the fluorescently labeled compound (in this case,the hybrid), as indicated by arrow 210. However, due to the flexiblenature of nucleic acids, as well as the only incremental increase insize of the hybrid over that of the target, this reduction may not besubstantial, and may not be easily detectable. In accordance with themethods of the present invention, however, this signal (P) iseffectively amplified by adding a polyionic compound 212 to the hybrid.Specifically, nucleic acids, i.e., the target sequence, are highlycharged species due to their negatively charged phosphate/sugarbackbones. Thus, this charge exists even when the nucleic acid exists indouble stranded form.

When a polyionic compound, e.g., polycation 212, i.e., polylysine, isadded to the hybrid 208, it associates with the hybrid 208, in anassociative complex 214, thereby substantially decreasing the rotationaldiffusion of the overall complex 214, as schematically illustrated byarrow 222. This difference is more readily detected.

In contrast, the reaction illustrated in panel II illustrates theinstance where the target sequence 202 does not include the sequence ofinterest that is complementary to the sequence of the fluorescent probe204. As such, the probe and target sequence are unable to hybridize, andthe rotational diffusion of the fluorescent component (the unhybridizedprobe) remains unchanged as illustrated by arrow 216. Further, when thepolycationic polyion is added to the reaction, it will again associatewith the highly charged target sequence as an associative complex 218.However, the polycation will not associate with the fluorescent probe204 due to the probe's uncharged character. As such, the rotationaldiffusion of the fluorescent compound (again the unhybridized probe 204)will remain unchanged, as illustrated by arrow 220. As a result, in thecase where hybridization occurs, i.e., the sequence of interest ispresent in the target, the fluorescence emissions from the reaction,when excited by polarized excitation light, is substantially polarizedas compared to that of the unhybridized probe. Conversely, where nohybridization occurs, i.e., the sequence of interest is not present,there is no change in the level of fluorescence polarization.Accordingly, a change in fluorescence polarization becomes an indicatorof the presence of the sequence of interest.

The methods and systems of the present invention are also useful incarrying out a variety of other binding assays, where the resultingcomplex has a substantially different charge from the charge of afluorescently labeled member of the binding pair. For example, in areceptor binding assay where a neutral, fluorescent ligand is bound tothe charged, unlabeled receptor, the methods of the present inventionserve to amplify a fluorescence polarization signal from the complex byassociating a large polyionic compound with that complex. Specifically,while the complex will, by itself, give a fluorescence polarizationresponse, as described herein, the complex with the associated polyioniccompound will be substantially greater. As will be appreciated, a widevariety of binding assays may be carried out in accordance with thepresent invention. Even a greater number of assays may be readilyconfigured to function in accordance with these methods, e.g., where thebound complex has a substantially different charge than a fluorescentlylabeled free member of the ultimate complex.

The methods and systems of the present invention also find particularusefulness in assaying for enzymatic activity where that activityproduces a product that has a substantially different charge than thesubstrate upon which the enzyme acted. One example of a class of enzymeassays that is suited for the methods and systems of the presentinvention are those that add or remove phosphate groups to or fromappropriate substrates, e.g. kinase and phosphatase assays. Interest inthese activities is substantial due to their roles in mediating a widevariety of biologically relevant response reactions in vivo. Inparticular, kinase and phosphatase reactions are often precursor, orintermediate signaling events in complex cellular behaviors such assurvival and proliferation. As such, their activities become ofparticular interest in addressing diseases where these behaviors aremalfunctioning, e.g., cancer, and the like.

As noted above, the present invention is particularly useful in assayingfor the activity of kinase enzymes. Kinase enzymes typically function byadding a phosphate group to a phosphorylatable substrate, e.g., protein,peptide, nucleoside, carbohydrate, etc. As phosphate groups are highlycharged, their addition to a particular substrate typically imparts asubstantial change in charge of the product over the substrate. As withthe assays described above, this change in charge in the product overthat of the substrate can be exploited by adding a polyionic compoundthat imparts a significant difference in the fluorescence polarizationof the product over the substrate.

Briefly, a phosphorylatable substrate is provided with a fluorescentlabeling group, as described above. The phosphorylatable substrate maybe neutral or it may be charged. Preferred substrates are neutral underthe relevant assay conditions. A variety of phosphorylatable substratesare commercially available. For example, rhodamine labeled substrate forprotein kinase A (PKA) is generally commercially available from PromegaInc., while other fluorescent phosphorylatable substrates may beobtained from Research Genetics, Inc.

Because the fluorescent phosphorylatable substrate is typicallyrelatively small, e.g., less than about 2 kD, it has a relatively highrate of rotational diffusion and thereby emits depolarized fluorescencewhen excited with polarized light. When contacted with a kinase enzymein the presence of a phosphate donor group, e.g., ATP, the substrate isphosphorylated, imparting two additional negative charges for eachphosphate incorporated. This net −2 charge, particularly in the case ofthe previously uncharged substrate, provides a basis for interaction ofthe product with a polyionic compound, e.g., a polycation such aspolylysine or polyhistidine. Once the polyion associates with thephosphorylated substrate, it significantly slows the rate of rotationaldiffusion, and thereby reduces the rate of fluorescence depolarization,e.g., increases the fluorescence polarization value. This change inpolarization is then detected and used to quantify the kinase reaction.

A schematic illustration of this reaction is shown in FIG. 3. As shown,the fluorescently labeled phosphorylatable substrate 302 is contactedwith a kinase enzyme 306, in the presence of phosphate 304, e.g., in theform of ATP. The reaction yields the phosphorylated product 308. Boththe fluorescent substrate 302 and the phosphorylated fluorescent product308 have relatively high rates of rotational diffusion due to theirsmall size. The fluorescent phosphorylated product is then contactedwith a polycation. Preferred polycations include polyamino acids such aspolylysine, polyhistidine or the like, with polyhistidine being mostpreferred. The polycation then associates with the negatively chargedphosphorylated fluorescent product, thereby drastically affecting itssize and rotational diffusion rate, which is then detected as describedrepeatedly herein. As will be appreciated, the polyionic component mayalternatively comprise a large molecule, e.g., a protein or the like,that has associated therewith multivalent metal cations selected from, eg., Fe³⁺, Ca²⁺, Ni²⁺ and Zn²⁺. Examples of such molecules include metalchelating proteins that chelate these ions, or the like. Specifically,these metal ions have relatively high affinity for oxygen, nitrogen andsulfur groups. As a result, they can impart a significant bindingaffinity to a large molecule (as a polyion) towards, e.g., phosphategroups in nucleic acids or phosphorylated substrates and the like, aswell as other groups bearing oxygen, nitrogen or sulfur groups, givingrise to the interaction that is used to significantly slow therotational diffusion rate of a fluorescent species, as described herein.

The present invention is also well suited to assay the reverse reaction.Specifically, the phosphatase reaction, which removes a phosphate groupfrom a phosphorylated substrate. This reaction follows substantially thereverse path of that shown in FIG. 3, and is schematically illustratedin FIG. 4. Briefly, the fluorescent phosphorylated compound 308, whichin this instance is the substrate, is contacted with the polycationiccompound 310 to yield the associative complex 312 where the polycationassociates with the charges imparted by the phosphate group. As notedabove with reference to FIG. 3, this complex has a slow rate ofrotational diffusion. When this complex is acted on by a phosphataseenzyme 414 it results in cleavage of the charged phosphate group and itsassociated polycation 404 from the fluorescent component 302, which inthis instance is the product. When free of the large polycationiccompound, the fluorescent product has a greatly increased rate ofrotational diffusion, e.g., emitting depolarized fluorescence. Again,this change in fluorescence polarization is detected as describedherein. As noted above, in some cases, the assay may preferably beperformed in a heterogeneous format, e.g., where the polyionic componentis added after the reaction of interest, in order to avoid any adverseeffects of the presence of the polyion on the reaction.

While the ability to perform a variety of assays is itself useful, thespecific applications to which these assays are put typically providesthe greatest value. Of particular interest is the ability to test theeffects of potential pharmaceutical candidate compounds on the variousactivities described above. Specifically, in pharmaceutical discoveryprocesses, large libraries of chemical compounds are generally screenedagainst pharmacologically relevant targets. These targets may includereceptors, enzymes, transporters, and the like. A variety of screeningassays and systems have been described. See, e.g., PublishedInternational Patent Application No. WO 98/00231, which is incorporatedherein by reference.

In brief, a particular reaction that is biologically or biochemicallyrelevant is carried out in the presence and absence of a compound thatis to be screened, and the effect of the compound is determined.Specifically, if the reaction is slowed or blocked by the presence ofthe test compound, then the compound is identified as an inhibitor ofthe reaction. Conversely where the reaction proceeds more rapidly or toa greater extent in the presence of the test compound, then the compoundis identified as an enhancer of the reaction. These screening assays arethen performed for a large number of different compounds, eitherserially or in parallel, in order to expedite the discovery of potentialeffectors of the reaction of interest.

III. Assay Systems

The present invention also provides assay systems that are used incarrying out the above-described methods. Typically, the assay systemsdescribed herein comprise a fluid receptacle into which the reagents areplaced for performing the assay. The fluid receptacle typicallycomprises a first reaction zone having disposed therein a first reagentmixture which comprises a first reagent having a fluorescent label, asecond reagent that reacts with the first reagent to produce afluorescently labeled product having a substantially different chargethan the first reagent and a polyionic compound.

FIG. 5 schematically illustrates an overall assay system for use inpracticing the present invention. Briefly, the overall system 500includes a reaction receptacle 502, as described above. A detector ordetection system 504 is disposed adjacent to the receptacle and withinsensory communication of the receptacle. The phrase “within sensorycommunication” generally refers to the detector that is positionedrelative to the receptacle so as to be able to receive a particularsignal from that receptacle. In the case of optical detectors, e.g.,fluorescence or fluorescence polarization detectors, sensorycommunication typically means that the detector is disposed sufficientlyproximal to the receptacle that optical, e.g., fluorescent signals aretransmitted to the detector for adequate detection of those signals.Typically this employs a lens, optical train or other detection element,e.g., a CCD, that is focused upon a relevant portion of the receptacleto efficiently gather and record these optical signals.

Detector 504 is typically connected to an appropriate data storageand/or analysis unit, e.g., a computer or other processor, which isgenerally capable of storing, analyzing and displaying the obtained datafrom the receptacle in a user comprehendible fashion, e.g., display 508.In certain embodiments, e.g., those employing microfluidic receptacles,the computer 506 is optionally connected to an appropriate controllerunit 510, which controls the movement of fluid materials within thechannels of the microfluidic device receptacle, and/or controls therelative position of the receptacle 502 and detector 504, e.g., via anx-y-z translation stage.

The receptacle also typically includes a detection zone as well as adetector disposed in sensory communication with the detection zone. Thedetector used in accordance with the present invention typically isconfigured to detect a level of fluorescence polarization of reagents inthe detection zone.

As used herein, the receptacle may take on a variety of forms. Forexample, the receptacle may be a simple reaction vessel, well, tube,cuvette, or the like. Alternatively, the receptacle may comprise acapillary or channel either alone or in the context of an integratedfluidic system that includes one or more fluidic channels, chambers orthe like.

In the case of a simple reaction vessel, well, tube, cuvette or thelike, the reaction zone and the detection zone typically refer to thesame fluid containing portion of the receptacle. For example, within thefluid containing portion of a cuvette, reagents are mixed, reacted andsubsequently detected. Typically, in order to expedite the process ofperforming assays, e.g., screening assays, multiplexed receptacles maybe used. Examples of such receptacles include, e.g., multiwell plates,i.e., 96-well, 384-well or 1536-well plates.

For capillary or channel based aspects, the reaction zone and thedetection zone may comprise the same fluid-containing portion of thereceptacle. However, in many aspects, the reaction zone and thedetection zone are separate fluid containing portions of the receptacle.Specifically, reagents may be mixed and reacted in one portion of thereceptacle, and subsequently moved to a separate detection zonewhereupon the reaction products, etc. are detected.

In particularly preferred aspects, the receptacle comprises amicrofluidic device. As used herein, the term “microfluidic device”refers to a device or body structure which includes and/or contains atleast one fluidic component, e.g., a channel, chamber, well or the like,which has at least one cross sectional dimension that is between about0.1 and about 500 μm, with these channels and/or chambers often havingat least one cross-sectional dimension between about 0.1 μm and 200 μm,in some cases between about 0.1 μm and 100 μm, and often between about0.1 μm and 20 μm. Such cross-sectional dimensions include, e.g., width,depth, height, diameter or the like. Typically, structures having thesedimensions are also described as being “microscale.” Microfluidicdevices in accordance with the present invention, typically include atleast one, and preferably more than one channel and/or chamber disposedwithin a single body structure. Such channels/chambers may be separateand discrete, or alternatively, they may be fluidly connected. Suchfluid connections may be provided by channels, channel intersections,valves and the like. Channel intersections may exist in a number offormats, including cross intersections, “T” intersections, or any numberof other structures whereby two channels are in fluid communication.

Because of their controllability, microfluidic device embodiments of thepresent invention are particularly useful in carrying out heterogeneousforms of the assays described herein. In particular, reactions areperformed in a first region of the microscale channel network. Theproducts of the reaction are then moved to a different portion of thechannel network, or additional components are brought into the originalportion of the channel network to mix with the products of the reaction.For example, the polyionic component of the assay methods describedherein can be added after the reaction of interest to ensure that itdoes not interfere with the reaction. Microfluidic systems provide theability to precisely move the various reagents through the variouschannels of the device, permitting their accurate measurement and timelyaddition. By way of a simple example, a phosphatase reaction may becarried out on a phosphorylated substrate in a first channel region of amicrofluidic device, yielding a phosphate group, e.g., ATP, and theunphosphorylated product, as well as unreacted substrate. The mixture isthen mixed with the polyionic component, e.g., polyhistidine, either bymoving the reaction mixture to a separate channel containing the polyionor by introducing the polyion into the reaction mixture in the originalchannel segment. The resulting mixture is then moved past a detectionpoint where the fluorescence polarization is measured.

The body structure of the microfluidic devices described hereintypically comprises an aggregation of two or more separate componentswhich when appropriately mated or joined together, form the microfluidicdevice of the invention, e.g., containing the channels and/or chambersdescribed herein. Typically, the microfluidic devices described hereinare fabricated as an aggregate of substrate layers. In particular, suchpreferred devices comprise a top portion, a bottom portion, and aninterior portion, wherein the interior portion substantially defines thechannels and chambers of the device.

FIG. 6 illustrates a two-layer body structure 610, for a microfluidicdevice. In preferred aspects, the bottom portion of the device 612comprises a solid substrate that is substantially planar in structure,and which has at least one substantially flat upper surface 614. Avariety of substrate materials may be employed as the bottom portion.Typically, because the devices are microfabricated, substrate materialswill be selected based upon their compatibility with knownmicrofabrication techniques, e.g., photolithography, wet chemicaletching, laser ablation, air abrasion techniques, injection molding,embossing, and other techniques. The substrate materials are alsogenerally selected for their compatibility with the full range ofconditions to which the microfluidic devices may be exposed, includingextremes of pH, temperature, salt concentration, and application ofelectric fields. Accordingly, in some preferred aspects, the substratematerial may include materials normally associated with thesemiconductor industry in which such microfabrication techniques areregularly employed, including, e.g., silica based substrates, such asglass, quartz, silicon or polysilicon, as well as other substratematerials, such as gallium arsenide and the like. In the case ofsemiconductive materials, it will often be desirable to provide aninsulating coating or layer, e.g., silicon oxide, over the substratematerial, and particularly in those applications where electric fieldsare to be applied to the device or its contents.

In additional preferred aspects, the substrate materials will comprisepolymeric materials, e.g., plastics, such as polymethylmethacrylate(PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™),polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone,polystyrene, polymethylpentene, polypropylene, polyethylene,polyvinylidine fluoride, ABS (acrylonitrile-butadiene-styrenecopolymer), and the like. Such polymeric substrates are readilymanufactured using available microfabrication techniques, as describedabove, or from microfabricated masters, using well known moldingtechniques, such as injection molding, embossing or stamping or thelike. Such polymeric substrate materials are preferred for their ease ofmanufacture, low cost and disposability, as well as their generalinertness to most extreme reaction conditions. Again, these polymericmaterials may include treated surfaces, e.g., derivatized or coatedsurfaces, to enhance their utility in the microfluidic system, e.g.,provide enhanced fluid direction, e.g., as described in U.S. Pat. No.5,885,470, which is incorporated herein by reference in its entirety forall purposes.

The channels and/or chambers of the microfluidic devices are typicallyfabricated into the upper surface of the bottom substrate or portion612, as microscale grooves or indentations 616, using the abovedescribed microfabrication techniques. The top portion or substrate 618also comprises a first planar surface 620, and a second surface 622opposite the first planar surface 620. In the microfluidic devicesprepared in accordance with the methods described herein, the topportion also includes a plurality of apertures, holes or ports 624disposed therethrough, e.g., from the first planar surface 620 to thesecond surface 622 opposite the first planar surface.

The first planar surface 620 of the top substrate 618 is then mated,e.g., placed into contact with, and bonded to the planar surface 614 ofthe bottom substrate 612, covering and sealing the grooves and/orindentations 616 in the surface of the bottom substrate, to form thechannels and/or chambers (i.e., the interior portion) of the device atthe interface of these two components. The holes 624 in the top portionof the device are oriented such that they are in communication with atleast one of the channels and/or chambers formed in the interior portionof the device from the grooves or indentations in the bottom substrate.In the completed device, these holes function as reservoirs forfacilitating fluid or material introduction into the channels orchambers of the interior portion of the device, as well as providingports at which electrodes may be placed into contact with fluids withinthe device, allowing application of electric fields along the channelsof the device to control and direct fluid transport within the device.

In many embodiments, the microfluidic devices will include an opticaldetection window disposed across one or more channels and/or chambers ofthe device. Optical detection windows are typically transparent suchthat they are capable of transmitting an optical signal from thechannel/chamber over which they are disposed. Optical detection windowsmay merely be a region of a transparent cover layer, e.g., where thecover layer is glass or quartz, or a transparent polymer material, e.g.,PMMA, polycarbonate, etc. Alternatively, where opaque substrates areused in manufacturing the devices, transparent detection windowsfabricated from the above materials may be separately manufactured intothe device.

As described in greater detail below, these devices may be used in avariety of applications, including, e.g., the performance of highthroughput screening assays in drug discovery, immunoassays,diagnostics, genetic analysis, and the like. As such, the devicesdescribed herein, will often include multiple sample introduction portsor reservoirs, for the parallel or serial introduction and analysis ofmultiple samples. Alternatively, these devices may be coupled to asample introduction port, e.g., a pipettor, which serially introducesmultiple samples into the device for analysis. Examples of such sampleintroduction systems are described in e.g., U.S. Pat. No. 5,779,868 andpublished International Patent Application Nos. WO 98/00705 and WO98/00231, each of which is incorporated herein by reference in itsentirety for all purposes. A schematic illustration of a microfluidicdevice incorporating an external sample pipettor is illustrated in FIG.7, described below.

Briefly, a microfluidic device 700, e.g., similar to that described withreference to FIG. 6, is provided having a body structure 702 whichincludes a network of internal channels 704 that are connected to aseries of reservoirs 706 disposed in the body structure 702. The variousreservoirs are used to introduce various reagents into the channels 704of the device. A capillary element 708 is coupled to the body structrure702, such that the channel 710 that is disposed within and runs thelength of the capillary element 708 is fluidly connected to the channelnetwork 704 in the body structure. This capillary element 708 is thenused to draw up a variety of different sample or test materials, inseries, for analysis within the device.

As described above, the methods and systems of the present inventiontypically rely upon a change in the level of fluorescence polarizationof the reaction mixture as a result of the reaction of interest. Assuch, an appropriate detection system is typically utilized todifferentiate polarized from depolarized emitted fluorescence. Generallyspeaking, such a detection system typically separately detectsfluorescent emissions that are emitted in the same plane of thepolarized excitation light, and fluorescent emissions emitted in a planeother than the plane of the excitation light.

One example of a detection system is shown in FIG. 8. As shown, thefluorescence polarization detector includes a light source 804, whichgenerates light at an appropriate excitation wavelength for thefluorescent compounds that are present in the assay system. Typically,coherent light sources, such as lasers, laser diodes, and the like arepreferred because of the highly polarized nature of the light producedthereby. The excitation light is directed through an optional polarizingfilter 806, which passes only light in one plane, e.g., polarized light.The polarized excitation light is then directed through an opticaltrain, e.g., dichroic mirror 810 and microscope objective 812 (andoptionally, reference beam splitter 808), which focuses the polarizedlight onto the sample receptacle (illustrated as a channel inmicrofluidic device 802), in which the sample to be assayed is disposed.

Fluorescence emitted from the sample is then collected, e.g., throughthe objective 812, and directed back through dichroic mirror 810, whichpasses the emitted fluorescence and reflects the reflected excitationlight, thereby separating the two. The emitted fluorescence is thendirected through a beam splitter 814 where one portion of thefluorescence is directed through an filter 816 that filters outfluorescence that is in the plane that is parallel to the plane of theexcitation light and directs the perpendicular fluorescence onto a firstlight detector 818. The other portion of the fluorescence is passedthrough a filter 820 that filters out the fluorescence that isperpendicular to the plane of the excitation light, directing theparallel fluorescence onto a second light detector 822. In alternativeaspects, beam splitter 814 is substituted with a polarizing beamsplitter, e.g., a Glan prizm, obviating the need for filters 816 and820. These detectors 818 and 822 are then typically coupled to anappropriate recorder or processor (not shown in FIG. 8) where the lightsignal is recorded and or processed as set out in greater detail below.Photomultiplier tubes (PMTs), are generally preferred as light detectorsfor the quantification of the light levels, but other light detectorsare optionally used, such as photodiodes, or the like.

The detector is typically coupled to a computer or other processor,which receives the data from the light detectors, and includesappropriate programming to compare the values from each detector todetermine the amount of polarization from the sample. In particular, thecomputer typically includes software programming which receives as inputthe fluorescent intensities from each of the different detectors, e.g.,for parallel and perpendicular fluorescence. The fluorescence intensityis then compared for each of the detectors to yield a fluorescencepolarization value. One example of such a comparison is given by theequation:

P=[I(∥)−I(⊥)]/[I(∥)+I(⊥)]C  (4)

as shown above, except including a correction factor (C), which correctsfor polarization bias of the detecting instrument. The computerdetermines the fluorescence polarization value for the reaction ofinterest. From that polarization value and based upon the polarizationvalues for free and bound fluorescence, the computer calculates theratio of bound to free fluorescence. Alternatively, the polarizationvalues pre and post reaction are compared and a polarization difference(ΔP) is determined. The calculated polarization differences may then beused as absolute values, e.g., to identify potential effectors of aparticular reaction, or they may be compared to polarization differencesobtained in the presence of known inhibitors or enhancers of thereaction of interest, in order to quantify the level of inhibition orenhancement of the reaction of interest by a particular compound.

FIG. 9 illustrates a flow-chart for the processes carried out by thecomputer using the above-described software programming. As shown, theprogrammed process begins at step 902 where the computer receives thefluorescence intensity data for the unreacted reagents in the reactionzone (e.g. in receptacle 502 of FIG. 5) from the two detectors, e.g.,detectors 818 and 820 of FIG. 8. The fluorescence polarization value (P)is then calculated in step 904, e.g., according to the equationsdescribed herein. At step 906, the computer receives fluorescenceintensity data for the reacted reagents from the two detectors. Again,at step 908, the P value is calculated for the reacted reagents. At step910, the P values for the reacted and unreacted reagents are compared,e.g., one is subtracted from the other to yield a ΔP value for thereaction. At this point, the ΔP value may be displayed as a measure ofthe reaction, e.g., its rate or completeness. Optionally, however, theΔP value may be compared to a standard ΔP value, i.e., from a reactionhaving a known rate, level of inhibition or enhancement, e.g., at step912. Through this comparison, the computer may then interpolate orextrapolate a quantitative measure of the reaction, its level ofinhibition or enhancement which quantitative measurement may then bedisplayed to the investigator, e.g., at step 914. As noted above, thecomputer may optionally include a determined polarization value forcompletely free and completely bound fluorescence. In that case,determination of fluorescence differences is not necessary, thuspermitting the omission of several steps of the program. In that case,the computer receives the fluorescence data from the detector for thereacted mixture. The computer then merely calculates the P value for thereaction mixture and determines the ratio of bound fluorescence to freefluorescence (e.g., in accordance with equation (3), supra). The ratiois then used to quantitate the reaction.

In the case of high-throughput screening assay systems, the computersoftware optionally instructs the correlation of a particular screenedresult to a particular sample or sample acquisition location. Thispermits the investigator to identify the particular reagents employed inany one assay.

FIG. 10 schematically illustrates a computer and architecture typicallyused in accordance with the present invention. In particular, FIG. 10Aillustrates an example of a computer system that may be used to executesoftware for use in practicing the methods of the invention or inconjunction with the devices and/or systems of the invention. Computersystem 1000 typically includes a display 1002, screen 1004, cabinet1006, keyboard 1008, and mouse 1010. Mouse 1010 may have one or morebuttons for interacting with a graphic user interface (GUI). Cabinet1006 typically houses a CD-ROM drive 1012, system memory and a harddrive (see FIG. 10B) which may be utilized to store and retrievesoftware programs incorporating computer code that implements themethods of the invention and/or controls the operation of the devicesand systems of the invention, data for use with the invention, and thelike. Although CD-ROM 1014 is shown as an exemplary computer readablestorage medium, other computer readable storage media, including floppydisk, tape, flash memory, system memory, and hard drive(s) may be used.Additionally, a data signal embodied in a carrier wave (e.g., in anetwork, e.g., internet, intranet, and the like) may be the computerreadable storage medium.

FIG. 10B schematically illustrates a block diagram of the computersystem 1000, described above. As in FIG. 10A, computer system 1000includes monitor or display 1002, keyboard 1008, and mouse 1010.Computer system 1000 also typically includes subsystems such as acentral processor 1016, system memory 1018, fixed storage 1020 (e.g.,hard drive) removable storage 1022 (e.g., CD-ROM drive) display adapter1024, sound card 1026, speakers 1028 and network interface 1030. Othercomputer systems available for use with the invention may include feweror additional subsystems. For example, another computer systemoptionally includes more than one processor 1014.

The system bus architecture of computer system 1000 is illustrated byarrows 1032. However, these arrows are illustrative of anyinterconnection scheme serving to link the subsystems. For example, alocal bus could be utilized to connect the central processor to thesystem memory and display adapter. Computer system 1000 shown in FIG.10A is but an example of a computer system suitable for use with theinvention. Other computer architectures having different configurationsof subsystems may also be utilized, including embedded systems, such ason-board processors on the controller detector instrumentation, and“internet appliance” architectures, where the system is connected to themain processor via an internet hook-up.

The computer system typically includes appropriate software forreceiving user instructions, either in the form of user input into setparameter fields, e.g., in a GUI, or in the form of preprogrammedinstructions, e.g., preprogrammed for a variety of different specificoperations. The software then converts these instructions to appropriatelanguage for instructing the operation of the optional materialtransport system, and/or for controlling, manipulating, storing etc.,the data received from the detection system. In particular, the computertypically receives the data from the detector, interprets the data, andeither provides it in one or more user understood or convenient formats,e.g., plots of raw data, calculated dose response curves, enzymekinetics constants, and the like, or uses the data to initiate furthercontroller instructions in accordance with the programming, e.g.,controlling flow rates, applied temperatures, reagent concentrations,etc.

As described above, the present invention is optionally carried out in amicrofluidic device or system. As such, it is generally desirable toprovide a means or system for moving materials through, between andamong the various channels, chambers and zones that are contained insuch devices. A variety of material transport methods are optionallyused if accordance with such microfluidic devices. For example, in onepreferred aspect material movement through the channels of a device iscaused by the application of pressure differentials across the channelsthrough which material flow is desired. This may be accomplished byapplying a positive pressure to one end of a channel or a negativepressure to the other end. In complex channel networks, controlled flowrates in all of the various interconnected channels may be controlled bythe inclusion of valves, and the like within the device structure, e.g.,to stop and start flow through a given channel. Alternatively, channelresistances may be adjusted to dictate the rate, timing and/or volume ofmaterial movement through different channels, even under a singleapplied pressure differential, e.g., a vacuum applied at a singlechannel port. Examples of such channel networks are illustrated in e.g.,U.S. patent application Ser. No. 09/238,467, filed Jan. 28, 1999, andU.S. Pat. No. 6,500,323 and 6,150,119, all of which are herebyincorporated herein by reference in their entirety for all purposes.

Alternately, for microfluidic applications of the present invention,controlled electrokinetic transport systems may be used. This type ofelectrokinetic transport is described in detail in U.S. Pat. No.5,858,195, to Ramsey, which is incorporated herein by reference for allpurposes. Such electrokinetic material transport and direction systemsinclude those systems that rely upon the electrophoretic mobility ofcharged species within the electric field applied to the structure. Suchsystems are more particularly referred to as electrophoretic materialtransport systems. Other electrokinetic material direction and transportsystems rely upon the electroosmotic flow of fluid and material within achannel or chamber structure which results from the application of anelectric field across such structures. In brief, when a fluid is placedinto a channel which has a surface bearing charged functional groups,e.g., hydroxyl groups in etched glass channels or glassmicrocapillaries, those groups can ionize. In the case of hydroxylfunctional groups, this ionization, e.g., at neutral pH, results in therelease of protons from the surface and into the fluid, creating aconcentration of protons at near the fluid/surface interface, or apositively charged sheath surrounding the bulk fluid in the channel.Application of a voltage gradient across the length of the channel, willcause the proton sheath to move in the direction of the voltage drop,i.e., toward the negative electrode.

“Controlled electrokinetic material transport and direction,” as usedherein, refers to electrokinetic systems as described above, whichemploy active control of the voltages applied at multiple, i.e., morethan two, electrodes. Rephrased, such controlled electrokinetic systemsconcomitantly regulate voltage gradients applied across at least twointersecting channels. In particular, the preferred microfluidic devicesand systems described herein, include a body structure which includes atleast two intersecting channels or fluid conduits, e.g., interconnected,enclosed chambers, which channels include at least three unintersectedtermini. The intersection of two channels refers to a point at which twoor more channels are in fluid communication with each other, andencompasses “T” intersections, cross intersections, “wagon wheel”intersections of multiple channels, or any other channel geometry wheretwo or more channels are in such fluid communication. An unintersectedterminus of a channel is a point at which a channel terminates not as aresult of that channel's intersection with another channel, e.g., a “T”intersection. In preferred aspects, the devices will include at leastthree intersecting channels having at least four unintersected termini.In a basic cross channel structure, where a single horizontal channel isintersected and crossed by a single vertical channel, controlledelectrokinetic material transport operates to controllably directmaterial flow through the intersection, by providing constraining flowsfrom the other channels at the intersection. For example, assuming onewas desirous of transporting a first material through the horizontalchannel, e.g., from left to right, across the intersection with thevertical channel. Simple electrokinetic material flow of this materialacross the intersection could be accomplished by applying a voltagegradient across the length of the horizontal channel, i.e., applying afirst voltage to the left terminus of this channel, and a second, lowervoltage to the right terminus of this channel, or by allowing the rightterminus to float (applying no voltage). However, this type of materialflow through the intersection would result in a substantial amount ofdiffusion at the intersection, resulting from both the natural diffusiveproperties of the material being transported in the medium used, as wellas convective effects at the intersection.

In controlled electrokinetic material transport, the material beingtransported across the intersection is constrained by low level flowfrom the side channels, e.g., the top and bottom channels. This isaccomplished by applying a slight voltage gradient along the path ofmaterial flow, e.g., from the top or bottom termini of the verticalchannel, toward the right terminus. The result is a “pinching” of thematerial flow at the intersection, which prevents the diffusion of thematerial into the vertical channel. The pinched volume of material atthe intersection may then be injected into the vertical channel byapplying a voltage gradient across the length of the vertical channel,i.e., from the top terminus to the bottom terminus. In order to avoidany bleeding over of material from the horizontal channel during thisinjection, a low level of flow is directed back into the side channels,resulting in a “pull back” of the material from the intersection.

In addition to pinched injection schemes, controlled electrokineticmaterial transport is readily utilized to create virtual valves whichinclude no mechanical or moving parts. Specifically, with reference tothe cross intersection described above, flow of material from onechannel segment to another, e.g., the left arm to the right arm of thehorizontal channel, can be efficiently regulated, stopped andreinitiated, by a controlled flow from the vertical channel, e.g., fromthe bottom arm to the top arm of the vertical channel. Specifically, inthe ‘off’ mode, the material is transported from the left arm, throughthe intersection and into the top arm by applying a voltage gradientacross the left and top termini. A constraining flow is directed fromthe bottom arm to the top arm by applying a similar voltage gradientalong this path (from the bottom terminus to the top terminus). Meteredamounts of material are then dispensed from the left arm into the rightarm of the horizontal channel by switching the applied voltage gradientfrom left to top, to left to right. The amount of time and the voltagegradient applied dictates the amount of material that will be dispensedin this manner. Although described for the purposes of illustration withrespect to a four way, cross intersection, these controlledelectrokinetic material transport systems can be readily adapted formore complex interconnected channel networks, e.g., arrays ofinterconnected parallel channels.

An example of a system employing this type of electrokinetic transportsystem in a microfluidic device, e.g., as illustrated in FIG. 7, isshown in FIG. 11. As shown, the system 1100 includes a microfluidicdevice 700, which incorporates an integrated pipettor/capillary element708. Each of the electrical access reservoirs 706, has a separateelectrode 1128-1136 disposed therein, e.g., contacting the fluid in thereservoirs. Each of the electrodes 1128-1136 is operably coupled to anelectrical controller 508 that is capable of delivering multipledifferent voltages and/or currents through the various electrodes.Additional electrode 1138, also operably coupled to controller 1108, ispositioned so as to be placed in electrical contact with the materialthat is to be sampled, e.g., in multiwell plate 502, when the capillaryelement 708 is dipped into the material. For example, electrode 1138 maybe an electrically conductive coating applied over capillary 708 andconnected to an electrical lead which is operably coupled to controller508. Alternatively, electrode 1138 may simply include an electrode wirepositioned adjacent the capillary so that it will be immersedin/contacted with the sample material along with the end of thecapillary element 708. Alternatively, the electrode may be associatedwith the source of material, as a conductive coating on the materialsource well or as a conductive material from which the source well wasfabricated. Establishing an electric field then simply requirescontacting the electrical lead with the source well material or coating.Additional materials are sampled from different wells on the multiwellplate 502, by moving one or more of the plate 502 and/or device 700relative to each other prior to immersing the pipettor 1138 into a well.Such movement is typically accomplished by placing one or more of thedevice 700 or multiwell plate 502 on a translation stage, e.g., theschematically illustrated x-y-z translation stage 1142.

In still a further optional application, hybrid material transportmethods and systems may be employed. Briefly, such systems often relyupon the use of electrokinetic forces to generate pressure differentialswithin microfluidic systems. Such hybrid systems combine thecontrollability of electrokinetic systems with the advantages ofpressure based systems, e.g., lack of electrophoretic biasing effects.Such hybrid systems are described in, e.g., Published InternationalPatent Application No. WO 99/16162, which is incorporated herein byreference in its entirety for all purposes.

A variety of other systems may be employed in practicing the presentinvention including without limitation, e.g., rotor systems, dipsticksystems, spotted array systems and the like.

IV. Kits and Reagents

The reagents for carrying out the methods and assays of the presentinvention are optionally provided in a kit form to facilitate theapplication of these assays for the user. Such kits also typicallyinclude instructions for carrying out the subject assay, and mayoptionally include the fluid receptacle, e.g., the cuvette, multiwellplate, microfluidic device, etc. in which the reaction is to be carriedout.

Typically, reagents included within the kit include the first reagentthat bears the fluorescent label, as well as the polyionic compound.These reagents may be provided in vials for measuring by the user, or inpre-measured vials or ampoules which are simply combined to yield anappropriate reaction mixture. The reagents may be provided in liquidand/or lyophilized form and may optionally include appropriate buffersolutions for dilution and/or rehydration of the reagents. Typically,all of the reagents and instructions are co-packaged in a single box,pouch or the like that is ready for use.

V. Examples

Example 1 Detection of Phosphorylated Product by FluorescentPolarization

An aliquot of a neutrally charged phosphorylatable substrate(Flourescein-QSPKKG-CONH₂) was incubated overnight with ATP and CDK2(cyclin dependent kinase). The mixture was analyzed by standardcapillary electrophoresis methods and showed complete conversion ofsubstrate to product. A negative control (no enzyme) was also prepared.The two reaction mixtures were diluted in 50 mM TAPS pH 9.0 buffer(1:40). The fluorescence polarization values were measured by excitingthe samples at 490 nm and measuring emitted fluorescence at 520 nm in acuvette of a fluorimeter equipped to measure fluorescence polarization.Aliquots of a poly-D-Lysine solution and water were added (each addedaliquot increased the poly-D-Lysine concentration by 6 μM). The resultsof the assay are illustrated in FIG. 12 which plots the fluorescentpolarization of the sample versus the amount of poly-D-lysine added.

As shown, the fluorescence polarization of both substrate (square) andproduct (diamond) in the absence of polylysine was about 38 millipolarization units (mP). Upon addition of polylysine, the fluorescencepolarization of the product increased significantly (to 72 and then to˜100 mP upon addition of a large excess of polylysine). The fluorescencepolarization of the substrate only increased to about 42 mP.

Example 2 Differentiation of Product Concentrations Using FluorescencePolarization

Additional experiments were carried out using poly-histidine in place ofpolylysine. In this case, the buffer used was 50 mM BisTris pH 6.5; themolecular weight of the polyhistidine used was 15800 daltons (availablefrom Sigma Chemical, St. Louis, Mo.).

Mixtures containing varying ratios of the substrates and products of twoserine/threonine kinases were prepared, CDK2 and Protein Kinase A (PKA).The CDK substrate was the same as that described for Example 1, above.The PKA substrate was: Fluor-LRRASLG where the C-terminus was either acarboxyl group or a carboxamide group. These mixtures were used asmodels for kinase reactions at varying degrees of substrate conversion.To these mixtures of substrate and product were added aliquots of apolyhistidine solution and water. The concentration of this aqueousstock was approximately 1.3 mM, and the final concentration was between10 and 25 mM.

Fluorescence polarization readings were again obtained by exciting at490 and detecting emitted fluorescence at 520 nm (both substrates werefluorescein labeled). FIGS. 13 and 14 show the results from theseexperiments. Briefly, FIGS. 13 and 14 are plots of fluorescencepolarization in increasing concentrations of phosphorylated product ascompared to substrate (denoted % conversion). In the case of FIG. 13,the substrate product are model substrate/products of a CDK2, while FIG.14 illustrates similar data for PKA substrate/product mixtures. As canbe seen, a very good linear dependence is observed between thefluorescence polarization signal and the percent conversion. Thus, themethod is well suited to follow the progress of kinase reactions andalso for the screening of chemical libraries for kinase inhibitors.

Example 3 Nucleic Acid Hybridization Assay Using FluorescencePolarization Detection

The assay methods were also employed in the detection of a nucleic acidhybridization reaction. This assay is particularly interesting due tothe lack of an immobilized target sequence that was to be interrogated.In particular, the entire assay was carried out in solution.

A fluorescein-labeled peptide nucleic acid molecule 202 was used inhybridization experiments with the DNA targets 192 and 182. PNAs aregenerally commercially available from the Applied Biosystems Division ofthe Perkin-Elmer Corporation (Foster City, Calif.). The sequence ofthese molecules is illustrated below. In the case of the PNA molecule,the given sequence illustrates the analogous sequence of a DNA molecule.The sequences of the three molecules are as follows:

202: 5′ Fl-O-GTCAAATACTCCA (SEQ. ID NO: 1) 192:5′ ATGGGCTGGAGTATTTGACCTAATT (SEQ. ID NO: 2) 182: 5′ CGCTGTGGATGCTGCCTGA(SEQ. ID NO: 3)

DNA sequence 192 contains 13 bases (shown in bold) that are fullycomplementary to the PNA probe 202, whereas 182 is a non-complementaryoligonucleotide.

A solution containing 1 μM of PNA 202 in 50 mM HEPES pH 7.5 was mixedwith either 5 μM of 192 or 5 μM of 182. The mixtures were left at roomtemperature for about 10 min. and then placed in the cuvette of afluorimeter equipped to measure fluorescence polarization. Themeasurements were carried out using 490 nm for excitation and 520 nm fordetection of fluorescence emission. The fluorescence polarization valueswere recorded first in the absence and then in the presence ofpoly-L-Lysine hydrobromide, having a molecular weight of approximately70-100 kD (Sigma Chemicals). The poly-L-Lysine was added from a stocksolution in water (approx. 440 μM). The final concentrations of the polyLysine were 4.4 and 8.8 μM. The resulting fluorescence polarizationvalues are shown in FIG. 15.

As can be seen, a polarization value of 86 mP was obtained for themixture containing 202 and the non-complementary 182. This increased to140 mP in the presence of poly-L-Lysine. In contrast, the 202 hybridwith the complementary 192 sequence showed a polarization value of about100 mP in the absence (not shown in FIG. 15) and 229 mP in the presenceof poly-L-Lysine. These results demonstrate that fluorescencepolarization in the presence of poly-L-Lysine can be used to detect theformation of specific PNA/DNA hybrids in solution. Such assays arereadily employed in detection of the presence or absence of a particularsequence within a sample, e.g., in sequencing by hybridization, sequencechecking, screening for sequence variants, e.g., polymorphisms, i.e.,SNPs, STRs, and the like.

Example 4 Detection of Single Nucleotide Substitution

The assay methods were employed to detect differential hybridization ofa nucleic acid probe to a perfectly complementary target sequence and atarget sequence incorporating a single base variation, e.g., a singlenucleotide polymorphism (SNP). In particular, a fluoresceinated PNAprobe having a sequence complementary to a subsequence of a targetsequence was used to probe the target including the subsequence and atarget in which an interior base of the subsequence was substituted fora different base.

The 5′ to 3′ sequences of the PNA probe (PNA 7637)(SEQ ID NO:4), thetarget DNA sequence (DNA 244)(SEQ ID NO:5) and single base mismatchtarget DNA sequence (DNA 245)(SEQ ID NO:6) were as follows:

PNA 7637: Fluorescein-CCTGTAGCA (SEQ ID NO: 4) DNA  244:TTGTTGCCAATGCTACAGGCATCGT (SEQ ID NO: 5) DNA  245: TTGTTGCCAATGCT GCAGGCATCGT (SEQ ID NO: 6)

The subsequence of the DNA targets complementary to the PNA probe are inbold. The position of the SNP is underlined.

The PNA probe 7637 was used at a final concentration of 250 nM, in areaction volume of 400 μl. The buffer used was 50 mM HEPES pH 7.5, 100mM NaCl. The solution also contained poly-L-Lysine at about 3 μM. One μlaliquots of the DNA targets 244 and 245 were added iteratively to thePNA solution and the fluorescence polarization (excitation 490, emission520 nm) was recorded after the addition of each aliquot. The data fromthis experiment is illustrated in FIG. 16. The perfectly complementarytarget/probe mixture (diamonds) showed substantially higher levels offluorescence polarization, than the single base mismatched mixture(squares), indicating that a higher level of hybridization had occurred.As can also be seen, hybridization plateaued at approximately a 1 Mexcess of target sequence in the perfectly complementary example.

Unless otherwise specifically noted, all concentration values providedherein refer to the concentration of a given component as that componentwas added to a mixture or solution independent of any conversion,dissociation, reaction of that component to a alter the component ortransform that component into one or more different species once addedto the mixture or solution. The method steps described herein aregenerally performable in any order unless an order is specificallyprovided or a required order is clear from the context of the recitedsteps. Typically, the recited orders of steps reflects one preferredorder.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. Although the present invention has beendescribed in some detail by way of illustration and example for purposesof clarity and understanding, it will be apparent that certain changesand modifications may be practiced within the scope of the appendedclaims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 6 <210> SEQ ID NO 1 <211> LENGTH: 13<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:  Peptide      Nucleic Acid Probe <400> SEQUENCE: 1 gtcaaatact cca              #                   #                   #      13 <210> SEQ ID NO 2<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence:Synthetic       Nucleic Acid <400> SEQUENCE: 2atgggctgga gtatttgacc taatt           #                  #               25 <210> SEQ ID NO 3 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence:  Synthetic       Nucleic Acid <400> SEQUENCE: 3cgctgtggat gctgcctga              #                  #                   # 19 <210> SEQ ID NO 4 <211> LENGTH: 9<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Peptide      Nucleic Acid Probe <400> SEQUENCE: 4 cctgtagca                #                   #                   #          9 <210> SEQ ID NO 5<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence:Synthetic       nucleic acids <400> SEQUENCE: 5ttgttgccaa tgctacaggc atcgt           #                  #               25 <210> SEQ ID NO 6 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:Synthetic      nucleic acids <400> SEQUENCE: 6ttgttgccaa tgctgcaggc atcgt           #                  #               25

What is claimed is:
 1. A method of determining whether a compound isphosphorylated, the compound comprising a fluorescent label, the methodcomprising: providing the compound in a mixture with a bindingcomponent, wherein the binding component comprises multivalent metalions and is sufficiently large to induce a shift in an amount ofpolarized fluorescence emitted from the compound, when the bindingcomponent binds the compound; and detecting whether the bindingcomponent binds to the compound by monitoring the amount of polarizedfluorescence emitted from the mixture, binding of the binding componentto the compound being indicative that the compound is phosphorylated. 2.The method of claim 1, wherein the binding component comprises apolymeric material having multivalent metal ions associated therewith.3. The method of claim 2, wherein the multivalent metal ions areselected from the group consisting of Fe³⁺, Ca²⁺, Ni²⁺ and Zn²⁺.
 4. Themethod of claim 2, wherein the multivalent metal ions are chelated tothe polymeric material.
 5. The method of claim 1, wherein the bindingcomponent is between 5 kD and 1000 kD.
 6. The method of claim 1, whereinprior to the providing step, the compound is contacted with an enzymethat either adds or removes a phosphate group to or from the compoundunder conditions suitable for addition or removal of a phosphate group,respectively.
 7. The method of claim 6, wherein prior to the providingstep, the compound is contacted with a kinase enzyme in the presence ofa phosphate donor group.
 8. The method of claim 6, wherein prior to theproviding step, the compound is contacted with an enzyme that removes aphosphate group from the compound.
 9. The method of claim 6, whereinprior to the providing step, the compound is contacted with an enzymethat either adds or removes a phosphate group to or from the compound,in the presence of at least a first test compound.
 10. The method ofclaim 1, wherein the compound comprises a polypeptide.
 11. An assaysystem, comprising: a first reaction vessel having disposed therein afirst reaction mixture comprising a phosphorylated first fluorescentcompound, and a binding component, wherein the binding componentcomprises multivalent metal ions and is sufficiently large to induce ashift in an amount of polarized fluorescence emitted by the compoundwhen bound by the binding component; and a fluorescence polarizationdetector positioned in sensory communication with the first mixture inthe reaction vessel.
 12. The assay system of claim 11, wherein thereaction vessel comprises a well in a multiwell plate.
 13. The assaysystem of claim 11, wherein the binding component comprises a polymericmaterial.
 14. The assay system of claim 11, wherein the bindingcomponent is between 5 kD and 1000 kD.
 15. The assay system of claim 11,wherein the multivalent metal ions are selected from the groupconsisting of Fe³⁻, Ca²⁻, Ni²⁻ and Zn²⁻.
 16. The assay system of claim15, wherein the binding component comprises chelating groups forchelating multivalent metal ions to the binding component.
 17. The assaysystem of claim 11, further comprising at least a second reaction vesselhaving a second mixture disposed therein, the second mixture comprisingan unphosphorylated first fluorescent compound and the bindingcomponent.
 18. The assay system of claim 11, wherein the first reactionmixture comprises an enzyme for adding a phosphate group to the firstfluorescent compound.
 19. The assay system of claim 11, wherein thefirst reaction mixture comprised an enzyme for removing a phosphategroup from a phosphorylated first compound.